Chronic infection forces T cells to differentiate into a state of exhaustion in which they can still recognize antigen but are unable to unleash antiviral agents and kill infected cells. High expression of the co-inhibitory molecule PD-1 is the hallmark of T cell exhaustion. Importantly, .PD-1 blockade results in the restoration of T cell effector functions to exhausted T cells. Presently, there are several outstanding questions regarding how PD-1 ligation alters a T cell: What are the factors recruited to the PD-1 cytoplasmic tail after engagement? Does PD-1 transmit the same signals in effector and exhausted T cells? How does PD-1 engagement affect the T cell's ability to generate polyfunctional responses? Can disruption of PD-1 signaling alter the progression to and susceptibility to T cell exhaustion? These are the questions that will be addressed in this application and the answers will shed light on how T cell exhaustion is enforced and how it can be overcome. Our central hypothesis is that PD-1 ligation induces distinct signals during various stages of T cell differentiation [(naive ->effector ->memory) versus (naive ->effector ->exhausted)]. Our overall approach is to study the effects of PD-1 signaling in both murine and human systems simultaneously, allowing us to exploit the advantages of each system to probe PD-1 function and decipher if there are any key differences. Aim 1 will examine the factors that are recruited to the PD-1 cytoplasmic tail in vitro and will ask how PD-1 engagement alters the generation of effector responses by employing novel reagents to engage PD-1 signaling pathways supplied by Core B and using state of the art imaging analysis provided by Core C. Aim 2 proposes to examine the effects of PD-1 signaling in vivo in order to better understand how PD-1 engagement leads to and contributes to T cell exhaustion. Using the well defined LCMV model system, we will test our hypothesis that distinct signaling complexes are recruited to PD-1 in exhausted T cells as compared to effector and memory T cells. Additionally, Core B will generate mice that will allow us to determine how alterations in PD-1 signaling affect the response to viral infection. Where appropriate, we will collaborate with the other projects in investigating the global signaling pathways altered by PD-1 ligation (Project 4) as well as the role exhaustion plays in controlling HIV disease (Project 1). Through these combined studies we expect to learn how PD-1 ligation blocks T cell activation, leads to the exhaustion phenotype, and uncover targets that will restore T cell function to chronic diseases such as HIV-1.